猪GDF11基因部分序列的克隆及组织表达分析

为了探讨生长分化因子11(Growth differentiation factor11,GDF11,又名BMP11)在胸腰椎数变异中的作用,本试验克隆了该基因包含外显子2在内的部分编码区,并进一步采用RT-PCR技术对其在猪胚胎和初生仔猪中的表达进行了分析。结果表明,在35d的猪胚胎中,后肢、牙龈、脑、肝脏、肾脏、胸椎、腰椎各组织均有明显的表达,而在前肢、眼、心脏、肺脏中的表达较弱,在颈椎和荐尾椎中没有观察到GDF11的表达。在45d猪胚胎的后肢、脑、眼、胸椎组织中GDF11的表达较强,而在前肢、牙龈、肺脏、肾脏、腰椎和荐尾椎的表达相对较弱,在肝脏中的表达极其微弱。在心脏和颈椎中没有检测到GDF11的表达。在55d的猪胚胎中,前肢、后肢、脑、眼、肝脏、颈椎、胸椎、腰椎组织中有明显的表达,肺脏和肾脏组织中的表达较强,牙龈和荐尾椎中的表达较弱,而在心脏中没有检测到GDF11的表达。3d仔猪的后肢、牙龈、脑、肾脏和腰椎组织中GDF11有明显的表达,脾脏组织的表达量较高,前肢、肝脏、心脏、背腰最长肌和肺脏中的表达相对较弱,在眼、颈椎和荐尾椎中的表达极弱,在胸椎中没有检测到表达。在所检测的不同时期的所有组织中,脑和肾脏组织表达明显地高于其他组织。     

Changes in mRNA of immune factors expressed by milk somatic cells of Holstein cows with hypocalcemia after calving

Changes in immune factors expressed by milk somatic cells from Holstein cows with hypocalcemia after calving were investigated in this study. Fourteen multiparous Holstein cows after their 3rd or 4th calving in one farm were used. The cows were divided into 2 groups: 7 cows needing treatment due to onset of hypocalcemia (hypocalcemia group; age = 5.53 ± 0.27 years, parity = 3.14 ± 0.14) and 7 cows without health problems (control group; age = 5.88 ± 0.31 years, parity = 3.57 ± 0.26). Milk samples were collected aseptically using a cannula and mRNA of immune factors expressed by milk somatic cells were analyzed. Milk samples (50 mL) were collected from the right rear mammary gland of cows before milking at day 1 and weeks 1, 2, 4, and 8 after calving. All milk samples showed a negative reaction to the California Mastitis Test. Levels of relative interleukin (IL)-6 and cathelicidin in the hypocalcemia group were lower than those in the control group in weeks 1 to 8. A significant difference in relative IL-6 levels was found in week 4 (P < 0.05). These results suggest that levels of IL-6 expressed by milk somatic cells may be affected by hypocalcemia in dairy cows.     

荷斯坦奶牛乳腺上皮细胞的体外培养

用外科手术的方法采取健康的泌乳期荷斯坦奶牛乳腺组织,采用机械剪切结合酶消化的方法,在体外进行原代乳腺上皮细胞分离培养。结果成功地培养出荷斯坦奶牛原代乳腺上皮细胞。显微镜下观察,细胞形成单层呈鹅卵石样;角蛋白免疫组化染色后,呈现阳性反应。经数次传代后,细胞增殖依旧旺盛。     

Bacterial lipopolysaccharide induces mRNA expression of an IkappaB MAIL through toll-like receptor 4

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein recently identified as a molecule appearing in immunocompetent organs after administration of bacterial lipopolysaccharide (LPS). Participation of Toll-like receptor (TLR) 4, which is a major form of LPS receptors, in the LPS-induced MAIL expression was investigated. When a human myelomonocytic cell line U937 was treated with phorbol 12-myristate 13-acetate for 3 days, the LPS-induced MAIL expression was much potentiated in parallel with an increase in TLR4 expression. The MAIL induction was attenuated when the cells were treated with a neutralizing antibody against TLR4. The in vivo induction of MAIL in the spleen was smaller in mice having a missense mutation of the Tlr4 gene than in normal control mice. These results collectively indicate that TLR4 contributes, at least in part, MAIL induction after LPS stimulation.     

Effects of chlorpromazine,pentoxifylline and dexamethasone on mRNA expression of lipopolysaccharide-induced inflammatory cytokines in bovine peripheral blood mononuclear cells

The effects of chlorpromazine (CPZ), pentoxifylline (PTX) and dexamethasone (DEX) on mRNA expression of lipopolysaccharide (LPS)-induced proinflammatory cytokines were examined in bovine peripheral blood mononuclear cells (PBMCs) in vitro. The expression of inflammatory cytokine mRNAs was analyzed by RT-PCR and Southern blot hybridization in bovine PBMCs. CPZ and DEX decreased the expression of cytokine mRNA (such as interleukin-1 beta and tumor necrosis factor-alpha) after stimulation with LPS in a dose-dependent manner. However, pretreatment with PTX had no inhibitory effect on the mRNA expression of proinflammatory cytokines. These results indicated that pretreatment with CPZ and DEX might be effective to reduce the production of LPS-induced inflammatory cytokines in bovine PBMCs in vitro.     

Effects of mycophenolic acid on inosine monophosphate dehydrogenase I and II mRNA expression in white blood cells and various tissues in sheep

Mycophenolic acid (MPA) is a mycotoxin commonly found as Penicillium genus secondary metabolite in feedstuffs and silages. Feeding with MPA contaminated silages may modulate the immune system in the farm animals and can cause appetite lost, ketosis, paralysis and abortion. The aim of the present study was to characterize the long-term MPA effect on both the inosine monophosphate dehydrogenase (IMPDH) isoforms I and II mRNA expression in white blood cells (WBC) and various tissue of healthy sheep. In treated animals 300 mg MPA/day/sheep was applied. In all investigated tissues the IMPDH I and II mRNA was abundant: WBC, spleen, thymus, ileum, jejunum, kidney, liver, pharyngeal and mesenterial lymph node. An efficiency-corrected relative quantification of the IMPDH types I and II isoforms mRNA were performed by normalizing with the constant reference gene expression of beta-actin. High IMPDH I mRNA expression levels were seen in kidney > mesenterial lymph node > jejunum > spleen > pharyngeal lymph node. Medium and low abundance was found in ileum > WBC > liver > thymus. Type II mRNA was highly expressed in liver > thymus > jejunum. In pharyngeal lymph node > spleen > ileum > mesenterial lymph node > kidney > WBC medium to low IMPDH II mRNA concentrations were detected. Under MPA treatment the IMPDH I mRNA expression was not significantly regulated in WBC, only trends of down- and upregulation were observed. Surprisingly in jejunum an upregulation could be observed (P < 0.05). In pharyngeal lymph node a tendency to downregulation was shown. This may be due to frequent ruminant activities and frequent exposition of MPA to the pharyngeal lymph nodes. In contrast to type I mRNA expression, IMPDH II mRNA was significantly downregulated in ileum (3.4-fold, P < 0.01) and tendencies in downregulation could be seen in jejunum (5.1-fold, P = 0.14). In addition, significant downregulation of IMPDH II gene expression over the entire feeding experiment could be shown in WBC of MPA-treated animals compared with untreated animals (P < 0.05). In conclusion, the recent study demonstrates that feeding sheep with MPA-contaminated silage did not induce IMPDH I mRNA expression in various tissues and blood, except in jejunum, but has suppressive effects on IMPDH II mRNA expression in WBC and ileum.     

荷斯坦奶牛妊娠早期血浆差异蛋白质组学研究

采用二维凝胶电泳(2-DE)结合MADIL-TOF-TOF串联质谱法对荷斯坦奶牛妊娠早期血浆进行蛋白质组学分析鉴定,发现了6种差异蛋白在妊娠后表达量上调,分别是凝集素1前体、转甲状腺素蛋白前体、3,5-二碘水杨酸络合的牛血清白蛋白的晶体结构的A链、白蛋白、未命名蛋白产物及结合珠蛋白-25同源物1前体。对这6种蛋白在不同时期含量的变化趋势及可能引起每种蛋白含量变化的原因和在妊娠过程中的功能进行了分析。     

荷斯坦牛中性粒细胞β-防御素基因克隆及其原核表达质粒的构建

根据已发表的牛β-防御素基因序列设计引物,从具有临床乳腺炎症状的荷斯坦奶牛血液中性粒细胞中提取RNA,克隆牛β-防御素基因并插入到原核表达载体pQE-30中,构建了原核表达质粒pQE-30/β-defesion。测序结果表明,克隆的目的基因长189bp,编码1个氨基酸,与已报道的牛β-防御素氨基酸序列同源性达95.6%。本研究为牛乳腺防御素的表达及功能、活性的研究奠定了一定的基础。     

Adhesion molecules and lymphocyte subsets in milk and blood of periparturient Holstein cows.

Migration of leukocytes into the mammary gland is an essential element of resistance to infection which is likely influenced by expression of adhesion molecules. The contribution of subsets to mammary gland resistance remains unclear. Mononuclear cells from milk and blood of dairy cows were examined for variation in CD4+, CD8+, and WC1+ (Workshop Cluster 1; marker for gammadelta T cells) lymphocyte phenotypes and expression of LFA-1 and L-selectin at several time points during the periparturient period and at Week 16 of lactation. Proportions of CD4+ T cells were higher (p < or = 10.05) in blood than milk at all times between Week 0 and Week 16 relative to calving; the inverse was true of CD8+ cells. Expression of L-selectin was lower (p < or = 0.05) on CD4+ cells and higher on CD8+ cells from milk. The WC1+ subset was more frequent in blood than in milk except at calving when the opposite was true. After calving, proportions of L-selectin+ WC1+ cells decreased steadily to Week 16. Expression of LFA-1 was examined on mononuclear cell populations and found to be lower on milk cells and did not vary over time. We conclude that proportions of T cells subsets differ significantly between blood and milk, particularly around calving. Corresponding variations in L-selectin expression may indicate a role for this molecule in regulating the movement of CD8+ and WC1+ T cells into the bovine mammary gland.     

体外培养奶牛乳腺上皮细胞的形态学观察

荷斯坦奶牛是我国饲养的主要奶牛品种。研究荷斯坦奶牛乳腺发育的生理、病理对于奶牛育种和保障奶牛健康均具有重大意义。因此，进行荷斯坦奶牛的乳腺上皮细胞分离培养及形态学研究，可为今后的工作奠定基础。本研究在形态学方面对培养的乳腺上皮细胞进行了系统的阐述，以期对今后的研究提供借鉴。     

Peripheral white blood cell counts throughout pregnancy in non-aborting Neospora caninum-seronegative and seropositive high-producing dairy cows in a Holstein Friesian herd

Pregnancy is characterized by transient changes in the maternal immune system, also evident at peripheral level. The present study analyzes the kinetics and possible factors affecting peripheral white blood cell populations throughout pregnancy in a herd of high-producing dairy cows chronically infected or not with Neospora caninum. We examined 54 pregnant parous cows: 29 Neospora-seronegative and 25 Neospora-seropositive cows. Blood samples were collected on Days 90, 120, 150, 180 and 210 of gestation. General Linear Model (GLM) repeated measures analysis of variance showed that the interaction Neospora-seropositivity × parity significantly affected total leukocyte, neutrophil and monocyte counts with lower levels of total leukocytes, lower neutrophil and higher monocyte counts recorded in primiparous Neospora-seropositive cows. In addition, N. caninum-seropositive cows had significantly increased monocyte counts on Day 180 of gestation compared to seronegative ones. Other factors significantly associated with changes in total and/or differential leukocyte profiles were period of pregnancy, season, twin pregnancy and milk production. In conclusion, a parity-associated effect of chronic N. caninum infection was observed on peripheral blood cell profiles in dairy cattle during gestation.     

奶牛S100A12基因克隆、原核表达及体外抗菌活性分析

克隆奶牛S100A12基因并在大肠杆菌中高效表达.采用RT-PCR扩增奶牛外周血白细胞中S100A12基因,构建原核表达载体pCold TF-S100A12,在大肠杆菌BL21(DE3)中IPTG诱导表达并纯化.SDS-PAGE和Westernblotting分析显示S100A12基因以融合蛋白形式表达,表达量占菌体总蛋白的46.7％,纯化的重组蛋白(80 mg/L).琼脂扩散法测定重组蛋白对乳腺炎主要致病菌大肠杆菌和金黄色葡萄球菌的抗菌活性.结果显示:重组蛋白对大肠杆菌有抗菌活性,而对金黄色葡萄球菌无抗菌活性.本研究为S100A12蛋白抗体制备及基因功能研究奠定了基础.     

Effects of body condition at far-off dry period on blood biochemistry,liver triacylglycerol and muscular monocarboxylate transporter-1 mRNA expression in tropical Holstein dairy cows during peripartum period

Effects of body condition score (BCS) at far-off dry period on blood biochemistry, liver triacylglycerol, and muscular monocarboxylate transporter-1 mRNA expression in tropical Holstein dairy cows during peripartum period were studied. Eight dry cows were divided into two groups of four cows based on their BCS at 8 weeks prepartum: high BCS (HBS) and low BCS (LBS). Blood samples were collected weekly from 8 weeks before expected calving date until 8 weeks postpartum for determination of serum glucose, non-esterified fatty acid (NEFA), and β-hydroxybutyrate (BHBA). Muscle and liver samples were biopsied at 2 weeks before expected calving date and at 2 weeks postpartum for determination of muscular monocarboxylate transporter-1 (MCT-1) mRNA expression and liver triacylglycerol concentrations. Decreased BCS and elevated serum NEFA and BHBA indicated that LBS cows entered to the NEB period earlier than did HBS cows. LBS cows showed lower liver TAG and higher muscular MCT-1 mRNA expression than did HBS cows. The MCT-1 mRNA expression at 2 weeks prepartum was negatively correlated with BCSs at 2–3 weeks prepartum and at 1–2 weeks postpartum. In conclusion, LBS cows most likely adapted to NEB by upregulation of muscular MCT-1 expression and had lower NEB consequences during postpartum period.     

牛肌肉生成抑制素基因功能区的克隆与原核表达

根据GenBank中牛肌肉生成抑制素（MSTN）基因序列设计了1对引物，在引物两端分别加EcoRⅠ和XhoⅠ识别位点。利用RT—PCR技术扩增出了牛MSTN功能区序列。分别构建克隆和原核表达载体，酶切、PCR鉴定及测序分析表明，该基因功能区序列的克隆载体和原核表达载体已成功构建。筛选阳性菌，经IPTG诱导，牛MSTN基因功能区在大肠埃希氏菌中成功表达。     

Association analysis between variants in bovine progesterone receptor gene and superovulation traits in Chinese Holstein cows

The objective of this study was to identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism. The PCR-RFLP method was applied to detect two reported single nucleotide polymorphisms (SNPs) of G59752C and T81637C (rs41614030) located in introns 3 and 4 of the bovine progesterone receptor (PGR) gene in 171 Chinese Holstein cows treated for superovulation and evaluate its associations with superovulation traits. In polymorphic locus 81637, all cows without superovulation response were g.81637TC and g.81637TT genotypes. Association analysis showed that these two SNPs had significant effects on the total number of ova (TNO) (p<0.05), and the T81637C polymorphism was significantly associated with the number of transferable embryos (p<0.05). In addition, significant additive effects (p<0.05) on TNO were detected in the polymorphisms of G59752C and T81637C. These results showed for the first time that the G59752C and T81637C polymorphisms in PGR gene were associated with superovulation traits and indicated that PGR gene can be used as a predictor for superovulation in Chinese Holstein cows.     

白三叶TrSAMDC1克隆及表达分析

S-腺苷甲硫氨酸脱羧酶(SAMDC)在植物抵御逆境胁迫中发挥着重要作用。通过分子生物学技术克隆得到一个全长为1559 bp的白三叶S-腺苷甲硫氨酸脱羧酶基因,并命名为TrSAMDC1。对该序列进行生物信息学分析表明:TrSAMDC1开放阅读框长度为1077 bp,编码358个氨基酸;预测其编码的蛋白为不稳定的亲水性蛋白,无跨膜结构,无信号肽,二级结构的主要构件为无规则卷曲,三级结构为同源二聚体,可能定位于细胞质中;系统进化树表明TrSAMDC1与其他豆科植物SAMDC亲缘关系很近,在进化上高度保守。分析该基因在重金属镉(CdSO₄)、低温(4 ℃)、高温(35 ℃)、干旱(PEG-6000)和盐(NaCl)等非生物胁迫以及100 μmol·L^-1脱落酸(ABA)和1 mmol·L^-1生长素(IAA)等激素处理下的表达模式发现TrSAMDC1的表达具有组织器官和时空特异性:所有处理都能显著上调叶片的相对表达量,并且在大多数处理12 h后达到峰值。而根系表达量虽较对照有差异,但对各种处理的敏感程度显著低于叶片。推测该基因能在调节植物的生长发育以及植物应对非生物胁迫尤其是高温和重金属镉胁迫中发挥重要作用,以上结果为进一步研究该基因奠定了基础。     

Collateral uterine blood flow in Holstein cows during the third trimester of pregnancy

Blood flow to the gravid and nongravid uterine horns of four multiparous Holstein cows (mean +/- SD, BW=641.8 +/- 95.4 kg; age=4.8 +/- 1.2 years; parity=3.0 +/- 1.2) was measured on days 225, 248, and 266 of gestation. Surgery was conducted on day 214.5 +/- 4.0 of gestation through the flank of the standing cows. Transit-time ultrasonic flow probes (diameter 12 or 14 mm) were fitted surgically around the uterine arteries of each cow. Surgery was completed within two hours of anesthesia, and the animals recovered rapidly following surgery. Uterine blood flow (UBF, l/min) was recorded at 10 sec intervals for approximately 23.5 hours; these values were averaged to determine UBF. The mean gravid UBF was significantly (P<0.05) greater than the nongravid UBF in this study. The range of the gravid and nongravid UBFs varied from 3.61 to 14.05 and 0.72 to 6.54 l/min, respectively. There were no changes (P>0.1) in the mean gravid and nongravid UBFs from day 225 to 266 of gestation.     

IGLL1基因在奶牛乳腺组织中的表达量差异分析

免疫球蛋白λ多肽1(immunoglobulin lambda-like polypeptide 1,IGLL1)是一种具有编码蛋白质特性的基因,位于17号染色体上。通过本实验室前期的奶牛乳腺组织转录组测序研究表明,IGLL1基因可能对奶牛乳脂的合成代谢产生影响。本试验以中国荷斯坦奶牛乳腺组织为研究对象,选取3头高脂中国荷斯坦奶牛,3头低脂中国荷斯坦奶牛的乳腺组织提取RNA并反转录为c DNA,进行RT-PCR,并对数据进行统计分析,研究IGLL1基因在奶牛乳腺组织中的表达差异。结果显示,该基因在高脂奶牛及低脂奶牛的乳腺组织中均有表达,且高脂奶牛的乳腺组织中的表达量显著高于低脂奶牛的表达量(P=0.0370.05)。研究表明,IGLL1基因可能对中国荷斯坦奶牛的乳脂的合成或代谢产生影响。该研究为奶牛的优良品种选育提供了试验基础。